This PPG addresses the earliest-known pathobiology in AD, its pathogenic significance, and its origins. We identified early and robust abnormalities of the endocytic-autophagic-lysosomal system (EALS) and have established direct links to AD-related genes, beta-amyloidogenesis and neurodegeneration. Using novel cell and mouse models that reproduce this early pathology with remarkable authenticity, we will define underlying mechanisms by applying state-of-the-art approaches ranging from single cell genomics to in vivo MR neuroimaging. The PPG has two cores (animal, analytical) and four projects. The first project examines the functional significance of abnormal endosomes, characterizes late endosomal pathology as a possible unifying link between endocytic and autophagic dysfunction in sporadic AD, trisomy 21 (Ts21), and PS-FAD, and studies the role of risk factors (App, ApoE, and cholesterol) in driving this pathology. The second project investigates the basis for impaired autophagy in AD, Ts21 and PS-FAD and an observed essential role of PS in autophagy-mediated protein degradation. The neuroprotective role of autophagy, the cell's mechanism for turnover of damaged organelles and inclusions, will be studied and strategies to modulate autophagy in vivo will be tested. The third project examines the importance of the endogenous cysteine protease inhibitor cystatin C in modulating endocytic and autophagic function and in protecting against the potentially cytotoxic consequences of lysosomal dysfunction and cathepsin imbalance as EALS pathology develops. The fourth project investigates genes associated with development of EALS dysfunction using single cell RNA microarray analysis in neurons in AD and Ts 21 brain before and as they develop endosomal pathology and when this pathology is reversed by a single change in App copy number in Ts21 models. Gene silencing will be used to identify new genes in Ts21 that promote endosomal and other AD pathology. These complementary studies will define comprehensively EALS pathobiology in AD and identify new therapeutic targets for early AD.